HIV-1 RT Research Articles

Nanogel-Conjugated Reverse Transcriptase Inhibitors and Their Combinations as Novel Antiviral Agents with Increased Efficacy against HIV-1 Infection.

Nucleoside reverse transcriptase inhibitors (NRTIs) are an integral part of the current antiretroviral therapy (ART), which dramatically reduced the mortality from AIDS and turned the disease from lethal to chronic. The further steps in curing the HIV-1 infection must include more effective targeting of infected cells and virus sanctuaries inside the body and modification of drugs and treatment schedules to reduce common complications of the long-term treatment and increase patient compliancy. Here, we describe novel NRTI prodrugs synthesized from cholesteryl-ε-polylysine (CEPL) nanogels by conjugation with NRTI 5'-succinate derivatives (sNRTI). Biodegradability, small particle size, and high NRTI loading (30% by weight) of these conjugates; extended drug release, which would allow a weekly administration schedule; high therapeutic index (>1000) with a lower toxicity compared to NRTIs; and efficient accumulation in macrophages known as carriers for HIV-1 infection are among the most attractive properties of new nanodrugs. Nanogel conjugates of zidovudine (AZT), lamivudine (3TC), and abacavir (ABC) have been investigated individually and in formulations similar to clinical NRTI cocktails. Nanodrug formulations demonstrated 10-fold suppression of reverse transcriptase activity (EC90) in HIV-infected macrophages at 2-10, 2-4, and 1-2 μM drug levels, respectively, for single nanodrugs and dual and triple nanodrug cocktails. Nanogel conjugate of lamivudine was the most effective single nanodrug (EC90 2 μM). Nanodrugs showed a more favorable pharmacokinetics compared to free NRTIs. Infrequent iv injections of PEGylated CEPL-sAZT alone could efficiently suppress HIV-1 RT activity to background level in humanized mouse (hu-PBL) HIV model.

NMR characterization of HIV-1 reverse transcriptase binding to various non-nucleoside reverse transcriptase inhibitors with different activities.

Human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) is an important target for antiviral therapy against acquired immunodeficiency syndrome. However, the efficiency of available drugs is impaired most typically by drug-resistance mutations in this enzyme. In this study, we applied a nuclear magnetic resonance (NMR) spectroscopic technique to the characterization of the binding of HIV-1 RT to various non-nucleoside reverse transcriptase inhibitors (NNRTIs) with different activities, i.e., nevirapine, delavirdine, efavirenz, dapivirine, etravirine, and rilpivirine. 1H-13C heteronuclear single-quantum coherence (HSQC) spectral data of HIV-1 RT, in which the methionine methyl groups of the p66 subunit were selectively labeled with 13C, were collected in the presence and absence of these NNRTIs. We found that the methyl 13C chemical shifts of the M230 resonance of HIV-1 RT bound to these drugs exhibited a high correlation with their anti-HIV-1 RT activities. This methionine residue is located in proximity to the NNRTI-binding pocket but not directly involved in drug interactions and serves as a conformational probe, indicating that the open conformation of HIV-1 RT was more populated with NNRTIs with higher inhibitory activities. Thus, the NMR approach offers a useful tool to screen for novel NNRTIs in developing anti-HIV drugs.

Novel Benzo[d]imidazole-based Heterocycles as Broad Spectrum Anti-viral Agents: Design, Synthesis and Exploration of Molecular Basis of Action

The design and synthesis of a novel series of benzo[d]imidazole-based heterocycles and their biological evaluation as antiviral agents are reported herein. 1-(1-Methyl-1H-benzo[d]imidazol- 2-yl)-2-thiocyanatoethanone 2 was used as a key intermediate for the synthesis of the thiazolylhydrazine 4, the thiazolylamine 5 and the methylthiazole 7. Coupling of compounds 5 or 7 with the appropriate diazotized aromatic amines gave the diazenyl derivatives 6a-c and 8a-c, respectively. The quinazoline derivative 12 was also synthesized. On the other hand, the phenylthio 20 and the phenylsulphonyl 22 bioisosteresand their respective diazenyl derivatives 21a-c and 23a-c were prepared. The synthesized compounds were evaluated for their HIV-1, HCV, SSPE and H1N1 inhibitory activities and were found to display very promising results. Furthermore, to investigate the underlying possible mechanism of action, in vitro and in silico screening of this series of benzo[d]imidazoles was performed against the viral enzymes HIV-1 RT, HCV NS3/4A serine protease and H1N1 NA1. Overall findings of the executed investigations highlight these novel compounds as very promising potent, broad spectrum antiviral agents.

Selection of 2'-deoxy-2'-fluoroarabinonucleotide (FANA) aptamers that bind HIV-1 reverse transcriptase with picomolar affinity.

Using a Systematic Evolution of Ligands by Exponential Enrichment (SELEX) protocol capable of selecting xeno-nucleic acid (XNA) aptamers, a 2′-deoxy-2′-fluoroarabinonucleotide (FANA) aptamer (referred to as FA1) to HIV-1 reverse transcriptase (HIV-1 RT) was selected. FA1 bound HIV-1 RT with KD,app values in the low pM range under different ionic conditions. Comparisons to published HIV-1 RT RNA and DNA aptamers indicated that FA1 bound at least as well as these aptamers. FA1 contained a 20 nucleotide 5′ DNA sequence followed by a 57 nucleotide region of FANA nucleotides. Removal of the fourteen 5′ DNA nucleotides did not affect binding. FA1's predicted structure was composed of four stems and four loops. All stem nucleotides could be modified to G-C base pairs (14 total changes) with a small effect on binding. Eliminating or altering most loop sequences reduced or abolished tight binding. Overall, results suggested that the structure and the sequence of FA1 were important for binding. FA1 showed strong inhibition of HIV-1 RT in extension assays while no specific binding to avian myeloblastosis or Moloney murine leukemia RTs was detected. A complete DNA version of FA1 showed low binding to HIV-1 RT, emphasizing the unique properties of FANA in HIV-1 RT binding.

Pronounced Inhibition Shift from HIV Reverse Transcriptase to Herpetic DNA Polymerases by Increasing the Flexibility of α-Carboxy Nucleoside Phosphonates.

Alpha-carboxynucleoside phosphonates (α-CNPs) are novel viral DNA polymerase inhibitors that do not need metabolic conversion for enzyme inhibition. The prototype contains a cyclopentyl linker between nucleobase and α-carboxyphosphonate and preferentially (50- to 100-fold) inhibits HIV-1 RT compared with herpetic DNA polymerases. A synthesis methodology involving three steps has been developed for the synthesis of a series of novel α-CNPs, including a Rh(II)-catalyzed O-H insertion that connects the carboxyphosphonate group to a linker moiety and an attachment of a nucleobase to the other end of the linker by a Mitsunobu reaction followed by final deprotection. Replacing the cyclopentyl moiety in the prototype α-CNPs by a more flexible entity results in a selectivity shift of ∼ 100-fold in favor of the herpetic DNA polymerases when compared to selectivity for HIV-1 RT. The nature of the kinetic interaction of the acyclic α-CNPs against the herpetic DNA polymerases differs from the nature of the nucleobase-specific kinetic interaction of the cyclopentyl α-CNPs against HIV RT.

Design, Synthesis, and Biological Evaluation of Novel 2-(Pyridin-3-yloxy)acetamide Derivatives as Potential Anti-HIV-1 Agents.

Through a structure-based molecular hybridization and bioisosterism approach, a series of novel 2-(pyridin-3-yloxy)acetamide derivatives were designed, synthesized, and evaluated for their anti-HIV activities in MT-4 cell cultures. Biological results showed that three compounds (Ia, Ih, and Ij) exhibited moderate inhibitory activities against wild-type (wt) HIV-1 strain (IIIB ) with EC50 values ranging from 8.18 μm to 41.52 μm. Among them, Ij was the most active analogue possessing an EC50 value of 8.18 μm. To further confirm the binding target, four compounds were selected to implement an HIV-1 RT inhibitory assay. In addition, preliminary structure-activity relationship (SAR) analysis and some predicted physicochemical properties of three active compounds Ia, Ih, and Ij were discussed in detail. Molecular docking studies were also carried out to investigate the binding modes of Ij and the lead compound GW678248 in the binding pocket of RT, which provided beneficial information for further rational design of non-nucleoside reverse transcriptase inhibitors.

QSAR models and scaffold-based analysis of non-nucleoside HIV RT inhibitors

A selection of 289 pyrimidine derivatives with anti-HIV RT activities as non-nucleoside HIV RT inhibitors (NNRTI) were studied. The associative neural network (ASNN) method was applied to develop a quantitative structure–activity relationship (QSAR) for anti-HIV RT activity. The calculated models were validated using the bagging approach. A consensus model with R2=0.87 and RMSE=0.5 was obtained from 10 individual models. Scaffold analysis and molecular docking of the compounds used in the QSAR model identified a potential chemical scaffold. The results showed that scaffold-based analysis of the QSAR model could be helpful in identifying potent scaffolds for further exploration than analyzing the overall model. Matched molecular pair analysis (MMPA) was applied in the QSAR model to characterize molecular transformations causing a significant change in the anti-HIV activity. The linear QSAR model was calculated to explore the structural features important for NNRTI activity. The results revealed that the activity of NNRT inhibitors is strongly dependent on their aromaticity and structural flexibility. The scaffold-based analysis of QSAR models with molecular docking and MMPA was found to be helpful in characterizing potential scaffolds for anti-HIV RT derivatives. The outcome of this study provides a deeper insight into the computer-aided scaffold-based design of novel molecules with HIV RT activities. It was also clearly shown that the consensus model's failure to correctly predict new chemical series could be due to the limitation of its applicability domain (AD). Redevelopment of models using new measurements can dramatically increase their AD and performance.

Investigation of the Dynamic Relaxation Behavior of Biomolecules Immobilized on Metal Electrode in Time Domain

Biomolecules such as DNA immobilized on metal electrodes and non-Faradaic current was measured in buffered solution. The metal-biomolecules-liquid-metal structure can be regarded as a liquid type of capacitor. The measurement of the capacitive current, which is affected by the ions, biomolecules, and liquid in the solutions. It is interesting to investigate the charging and discharging process in time domain, which brings important information of the dynamic behavior of the whole system. In this study, double-stranded DNA (dsDNA) was immobilized on gold electrodes and the capacitive current was measured in buffer solution with a pulse stimulus. Different relation time constants were revealed for various surface modifications. The transient current of the sensor was measured in de-ionized water, TE buffer before and after dsDNA immobilized, different concentrations of HIV-1 RT protein. This technique shows ultra-high sensitivity of the protein detection (1 fM), indicating that this technique is promising for biosensor applications.

A novel family of diarylpyrimidines (DAPYs) featuring a diatomic linker: Design, synthesis and anti-HIV activities

To improve the conformational flexibility and positional adaptability of the traditional diarylpyrimidines (DAPYs), a family of diarylpyrimidines featuring a C–N diatomic linker between the left wing benzene ring and the central pyrimidine was firstly designed, synthesized, and evaluated for in vitro anti-HIV activity. Most of target molecules showed excellent activities against wild-type (WT) HIV-1. Among them the most potent two compounds 12h and 12r displayed extremely potent WT HIV-1 inhibitory activities with an EC50 of 2.6nM and 3.0nM, respectively, while their selective index (CC50/EC50) values were both over 1000. Another compound 12b (EC50 14.9nM) was also noteworthy due to its high SI of 18,614. Moreover, all of compounds were evaluated for their WT HIV-1 reverse transcriptase activities, which shown that the newly synthesized CH2NH-DAPYs bind to HIV-1 RT and belong to the genuine NNRTIs. However, the synthesized compounds lack the activities against HIV-1 double mutant (RES056) and HIV-2 (ROD). Thus it is an upcoming objective to improve the activities against HIV-1 double mutants.

Differential regulatory activities of viral protein X for anti-viral efficacy of nucleos(t)ide reverse transcriptase inhibitors in monocyte-derived macrophages and activated CD4+ T cells

Vpx encoded by HIV-2 and SIVsm enhances retroviral reverse transcription in macrophages in vitro by mediating the degradation of the host SAMHD1 protein that hydrolyzes dNTPs and by elevating cellular dNTP levels. Here we employed RT-SHIV constructs (SIV encoding HIV-1 RT) to investigate the contribution of Vpx to the potency of NRTIs, which compete against dNTPs, in monocyte-derived macrophages (MDMs) and activated CD4+ T cells. Relative to HIV-1, both SIV and RT-SHIV exhibited reduced sensitivities to AZT, 3TC and TDF in MDMs but not in activated CD4+ T cells. However, when SIV and RT-SHIV constructs not coding for Vpx were utilized, we observed greater sensitivities to all NRTIs tested using activated CD4+ T cells relative to the Vpx-coding counterparts. This latter phenomenon was observed for AZT only when using MDMs. Our data suggest that Vpx in RT-SHIVs may underestimate the antiviral efficacy of NRTIs in a cell type dependent manner.

Enzyme Selectivity of HIV Reverse Transcriptase: Conformations, Ligands, and Free Energy Partition

Atomically detailed simulations of HIV RT are performed to investigate the contributions of the conformational transition to the overall rate and specificity of enzyme catalysis. A number of different scenarios are considered within Milestoning theory to provide a more complete picture of the process of opening and closing the enzyme. We consider the open to closed transition in the absence of and with the correct and incorrect substrates. We also consider the free energy profile and the kinetics of the conformational change after the chemistry step in which a new base was added to the DNA, but the DNA was not yet displaced. We partition the free energy along the reaction coordinate and analyze the importance of different protein domains. Strikingly, significant influence on the free energy profile is detected for amino acids far from the active site. The overall long-range impact is about 50 percent of the total. We also illustrate that the overall rate is not necessarily determined by the highest free energy barrier along the reaction path (with respect to the free enzyme and substrate) and that the specificity is not necessarily determined by the same reaction step that determines the rate.

Synthesis and molecular docking studies of oxochromenyl xanthenone and indolyl xanthenone derivatives as anti-HIV-1 RT inhibitors.

A series of novel oxochromenyl xanthenone and indolyl xanthenone derivatives were obtained by one-pot reaction of substituted salicylaldehyde, 4-hydroxy coumarin/indole and dimedone at ambient temperature condition using eco-friendly reusable ionic liquid [Hmim]HSO4 in ethanol solvent. Excellent yields, mild reaction condition, and simple experimental work-up procedure are some of the advantages of this method. The obtained derivatives were studied for their molecular docking as an anti-HIV-1 RT. All synthesized compounds from indolyl xanthenone and chromenyl xanthenone series were be docked into the non-nucleoside inhibitor binding pocket (NNIBP) of HIV-1 RT. Compounds 4r, 4j and 4k was found to be good around -12.487, -12.457, -12.256, respectively, with respective to native ligand TMC278, was found to be -13.413 which confirms that these compounds might have potent RT inhibition activity.

Investigation of the Dynamic Relaxation Behavior of Biomolecules Immobilized on Metal Electrode in Time Domain

Biomolecules such as DNA immobilized on metal electrodes and non-Faradaic current was measured in buffered solution. The metal-biomolecules-liquid-metal structure can be regarded as a liquid type of capacitor. The measurement of the capacitive current, which is affected by the ions, biomolecules, and liquid in the solutions. It is interesting to investigate the charging and discharging process in time domain, which brings important information of the dynamic behavior of the whole system. In this study, double-stranded DNA (dsDNA) was immobilized on gold electrodes and the capacitive current was measured in buffer solution with a pulse stimulus. Different relation time constants were revealed for various surface modifications. The transient current of the sensor was measured in de-ionized water and in 30 mM TE buffer before dsDNA immobilized metal electrode, and in 30 mM TE buffer after dsDNA immobilized on the metal electrode, followed by different concentrations of HIV-1 RT protein, ranging from 1 aM to 100fM. This technique shows ultra-high sensitivity of the protein detection (1 aM), indicating that this technique is promising for biosensor applications. Figure 1 shows the schematic of device for capacitive current measurements.

Fragment screening identifies novel HIV-1 RT inhibitors

Fragment screening identifies novel HIV-1 RT inhibitors

Synthesis, Anti-HIV, Antimicrobial Evaluation and Structure Activity Relationship Studies of Some Novel Benzimidazole Derivatives

In this study 26 novel benzimidazole compounds bearing alkyl chain as linker at N-1 position were synthesized and evaluated to investigate their possible anti-HIV and antimicrobial activities. Structures of the synthesized compounds were elucidated by spectral data. HIV-1 RT inhibitory activity was evaluated by using HIV-1 RT RNA-dependent DNA polymerase activity assay. Among these derivatives, compounds 43, 45, 50 and 51 were found to have reasonable HIV-1 RT inhibitory activity while the rest exhibited weak activity in comparison to the standard efavirenz. On the other hand, most of the test compounds were found to be significantly effective against gram positive bacteria (Staphylococcus aureus and Bacillus subtilis) and some gram-negative bacterial strains (Escherichia coli, Salmonella typhi, Klebsiella pneumoniae and Pseudomonas aeruginosa), among which compounds 37, 43, 45, 50 and 51 surfaced out as the most effective antibacterials but less effective than the standard ciprofloxacin. Compounds 33, 41 and 47 also showed significant activity against almost all the bacterial strains while all other compounds showed moderate activity towards all the bacterial strains. In case of fungal strains like Candida albicans and Aspergillus niger, almost all the compounds were found to exhibit potency comparable or more than that of standard drug fluconazole except for the compounds 34, 39, 42, 47 and 48 which exhibited moderate to good activity against both the fungal strains. A structure activity relationship (SAR) as well as virtual ADME and toxicity prediction were carried out and a connection between activities, electronic, physicochemical properties and toxicity levels of the target compounds was determined. Keywords: ADME, antibacterial activity, antifungal activity, anti-HIV activity, benzimidazole, SAR, toxicity prediction.

Detection of HIV-1 RT Protein Using AlGaN/GaN High Electron Mobility Transistors

As AIDS caused by (human immunodeficiency virus type 1) has now spread to every country in the world. Statistics show that approximately 40 million people are currently living with HIV infection, and an estimated 35 million have died from this disease since the beginning of the epidemic. In this study, we use DNA-immobilized AlGaN/GaN high electron mobility transistors (HEMTs) for detecting low concentration of HIV-1 RT protein (1 fM) binding with DNA electronically, showing that this technique is promising for biosensor applications. Figure 1 (a) and (b) show the schematic of RT immobilized HEMT and the top-view of the device respectively. The HEMT structure consisted of a 3 μm-thick GaN buffer, 150 Å-thick undoped Al0.25Ga0.75N and 10 Å-thick undoped GaN cap layer. Mesa isolation was formed by Inductively Coupled Plasma (ICP) etching system for 28 seconds. Ti, Al, Ni and Au were deposited by Electron Beam Evaporation and were annealed at 850 °C for 45 seconds under N2 flowing to form ohmic contacts. The source and drain of HEMT were isolated by photoresist except the gate metal region. The DNA was immobilized on the gate region for 24 hours followed by surface blocking with bovine serum albumin (BSA). The HIV-1 RT protein solutions were then added on the sensing region of the sensor, and then waited for 30 minute for binding. After HIV-1 RT protein binding with DNA, the sensor was washed with 1X PBS to remove the unbound HIV-1 RT protein. 1X PBS was added on the sensor for electrical measurements. The sensor was measured by applying a dc bias of drain-source voltage (Vds) at 0.5V first, and then the gate voltage (Vg) was applied 0.5 V on the gate electrode. The drain current (Id) of the transistor was recoded in real-time when the drain-source voltage and the gate voltage were applied. The total charge was calculated by integrating the drain currents with the increasing time. Due to calculation of the total charges, any tiny difference in drain currents resulted from different HIV-1 RT protein concentrations may cause a significantly huge difference in the total charges. The total charge counted at a certain time for various concentrations of HIV-1 RT protein is shown in figure 2. It is obviously seen that the difference in total charges among different surface modification. Higher HIV-1 RT protein concentrations, including 1fM, 10fM, 100fM and 1pM were tested and caused more accumulated charges. Therefore, the change in total charge with varying concentration of HIV-1 RT protein can represent sensor calibration curve. The detection limit was then determined to be 1fM for HIV-1 RT protein measurement. The high sensitivity and the low detection limit of the sensor resulted from the charge accumulation and the less screening effect during the dynamic change of the drain current. In the detection, only a small amount of sensing materials is required and a short detection time is taken. The developed system has a great potential to measure an extremely low concentration of different proteins for biomedical applications. Figure 1

Detection of HIV-1 RT Protein Using AlGaN/GaN High Electron Mobility Transistors

As AIDS caused by (human immunodeficiency virus type 1) has now spread to every country in the world. Statistics show that approximately 40 million people are currently living with HIV infection, and an estimated 35 million have died from this disease since the beginning of the epidemic1. In this study, we use DNA-immobilized AlGaN/GaN high electron mobility transistors (HEMTs) for detecting low concentration of HIV-1 RT protein (1 fM) binding with duplex DNA (dsDNA) electronically, showing that the technique is promising for biosensor applications.

Anti-HIV diarylpyrimidine–quinolone hybrids and their mode of action

A molecular hybridization approach is a powerful tool in the design of new molecules with improved affinity and efficacy. In this context, a series of diarylpyrimidine–quinolone hybrids were synthesized and evaluated against both wt HIV-1 and mutant viral strains. The most active hybrid 5a displayed an EC50 value of 0.28±0.07μM against HIV-1 IIIB. A couple of enzyme-based assays clearly pinpoint a RT-targeted mechanism of action. Docking studies revealed that these hybrids could be well located in the NNIBP of HIV-1 RT despite the bulky and polar properties of a quinolone 3-carboxylic acid moiety in the molecules.

Synthesis and Anti Hiv-1 Reverse Transcriptase Evaluation of A Series of N-Mono Substituted Thiourea Derivatives

Thirteen kinds of N-monosubstituted thioureas have been synthesized from various primary amines through three differ- ent methods. The chemical structures of all the compounds have been characterized by the various spectral analyses. Four of them were evaluated for the anti-HIV-1 activity. The results showed that compound 1b, showing the IC 50 = 29.7 (μg/ mL) to the strain of ROD of HIV-1, CC 50 > 50 (μg/mL), SI (selectivity index) > 2, was the best one among the test com- pounds. As for other compound 1a, 1c and 1d, the SI of them was less than 1, which means that these compounds might be toxic at the therapeutic level. Both the steric, electronic and topologic descriptors of the molecules were calculated to assist understanding the basic relationship between the structure and the biological activity. The docking result of 1c with HIV-1 reverse transcriptase (HIV-1 RT, PDB ID: 2HNZ) showed that there were still more unexploited rooms in the active site of the binding pocket of HIV-1 RT with compounds 1c

Enzymatic synthesis of acyclic nucleoside thiophosphonate diphosphates: Effect of the α-phosphorus configuration on HIV-1 RT activity

The acyclic nucleosides thiophosphonates (9-[2-(thiophosphonomethoxy)ethyl]adenine (S-PMEA) and (R)-9-[2-(thiophosphonomethoxy)propyl]adenine (S-PMPA), exhibit antiviral activity against HIV-1, -2 and HBV. Their diphosphate forms S-PMEApp and S-PMPApp, synthesized as stereoisomeric mixture, are potent inhibitors of wild-type (WT) HIV-1 RT. Understanding HIV-1 RT stereoselectivity, however, awaits resolution of the diphosphate forms into defined stereoisomers. To this aim, thiophosphonate monophosphates S-PMEAp and S-PMPAp were synthesized and used in a stereocontrolled enzyme-catalyzed phosphoryl transfer reaction involving either nucleoside diphosphate kinase (NDPK) or creatine kinase (CK) to obtain thiophosphonate diphosphates as separated isomers. We then quantified substrate preference of recombinant WT HIV-1 RT toward pure stereoisomers using in vitro steady-state kinetic analyses. The crystal structure of a complex between Dictyostelium NDPK and S-PMPApp at 2.32Å allowed to determine the absolute configuration at the α-phosphorus atom in relation to the stereo-preference of studied enzymes. The RP isomer of S-PMPApp and S-PMEApp are the preferred substrate over SP for both NDPK and HIV-1 RT.